A molecular module related to the abundance of oyster kati2 protein and high temperature resistance and application thereof

Abstract

The application discloses a molecular module related to oyster KAT2 protein abundance and high-temperature resistance and application thereof, and belongs to the field of genetic engineering and aquatic genetic breeding. The molecular module comprises three SNP markers of a KAT2 gene coding region, namely, c.243 C>T, c.507 A>G and c.801 C>A. The application also discloses primer pairs for detecting the molecular module and application thereof in high-temperature breeding of oysters. The oysters with a genotype combination of TT / GG / AA at the three sites have a significantly higher KAT2 protein expression abundance and survival rate under high-temperature stress than other genotypes. The application can realize accurate evaluation by detecting only three SNP sites, has low typing cost and reliable results, and is suitable for molecular marker assisted selection of high-temperature resistant strains of oysters.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and aquatic genetic breeding, and in particular to a molecular module related to the abundance of KAT2 protein in oysters and its application. Background Technology

[0002] Oysters are among the most productive farmed marine shellfish globally, playing a vital role in the stability of marine ecosystems and coastal fisheries. However, in recent years, continuous global warming and frequent extreme heat events in summer have led to large-scale outbreaks of mortality in farmed oysters, becoming a primary environmental factor restricting the healthy and sustainable development of the oyster industry. Existing physiological and ecological studies have clearly shown that high-temperature stress directly disrupts the energy metabolism balance of oyster cells, triggering a cascade of oxidative stress and apoptosis, ultimately leading to individual death. To address this breeding bottleneck, the industry has attempted to use conventional methods such as hybridization breeding and phenotypic selection to screen for heat-resistant strains. However, these methods heavily rely on in vivo heat resistance challenge tests, which are not only time-consuming and costly, but also subject to the interactive influence of multiple environmental factors such as seawater temperature, salinity, and food abundance, making it difficult to guarantee phenotypic repeatability and identification accuracy, significantly hindering the efficient breeding process of heat-resistant varieties.

[0003] With the rapid development of molecular biology and high-throughput sequencing technologies, genome-wide selection (GS) breeding has demonstrated its potential to improve the breeding efficiency of complex traits in aquaculture species such as oysters and scallops. However, the implementation of GS breeding strategies relies on high-density molecular markers across the entire genome, making genotyping extremely costly. This is especially true for oysters, which exhibit high genetic diversity and rapid linkage disequilibrium decay. The number of markers required to achieve reliable prediction accuracy further increases, making the breeding costs prohibitive for industrial application. Therefore, a key direction in current aquaculture molecular breeding research is to significantly reduce the scale of genotyping markers while maintaining selection accuracy, establishing a low-cost, high-efficiency targeted genetic improvement technology system. In this regard, molecular module breeding strategies based on functional site integration, by focusing on key regulatory nodes of target traits, are expected to avoid redundant information from scattered markers across the entire genome. However, in the area of ​​oyster stress resistance traits, especially functional molecular modules related to high-temperature resistance, systematic localization and functional validation studies are still lacking, leaving related breeding applications largely unexplored.

[0004] Oyster ( Crassostrea gigas ) and Fujian oyster ( Crassostrea angulataThe *Crassostrea gigas* and *Crassostrea kwangsiensis* are the most closely related sister species in the genus *Crassostrea gigas*, and artificial hybridization between the two species is feasible. They have already shown significant differentiation in temperature adaptability: the half-lethal temperature and maximum basal metabolic rate of the *Crassostrea gigas* are significantly higher than those of the *Crassostrea gigas*, indicating that the former has stronger heat tolerance potential. Comparative transcriptomic and proteomic studies further revealed systematic differences in acetylation modification levels in energy metabolism pathways between the two species under high-temperature stress. Specifically, the protein abundance of the core acetyltransferase KAT2 in the *Crassostrea gigas* exhibits constitutive high expression, and its expression differences are closely related to the acetylation modification status of key enzymes in glycolysis and the tricarboxylic acid cycle. However, current understanding of the genetic regulatory mechanism of KAT2 protein abundance is extremely limited. There is a lack of analysis of the causal relationship between upstream genomic sequence variations and protein expression levels, and no research reports on its use as a quantifiable molecular phenotype for screening heat tolerance-related genetic markers. This knowledge gap makes it difficult to directly translate the KAT2-mediated metabolic-epigenetic regulatory axis into an operational breeding tool. Therefore, it is urgent to systematically identify the key genetic loci regulating the abundance of KAT2 protein and the molecular modules they constitute at the genomic level, so as to provide usable functional elements and marker resources for the precise molecular design breeding of oyster heat resistance traits. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular module and its application related to the abundance of KAT2 protein and high-temperature resistance in oysters, thereby addressing the problems existing in the prior art. The molecular module provided by this invention only requires detection of three SNP loci to accurately assess the abundance of KAT2 protein and high-temperature resistance in oysters, significantly reducing genotyping costs compared to genome-wide selection breeding. Validation in wild and hybrid populations showed that individuals with the dominant TT / GG / AA genotype exhibited significantly higher KAT2 expression levels and higher survival rates after heat shock, providing an economical, efficient, and reliable genetic tool for molecular breeding of heat-resistant oysters.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a molecular module related to the abundance of KAT2 protein in oysters and high-temperature resistance, the molecular module comprising a first SNP molecular marker, a second SNP molecular marker, and a third SNP molecular marker; The first SNP is marked by a C / T mutation at the 51st base of the sequence shown in SEQ ID NO.1; The second SNP molecular marker is an A / G mutation at the 1265th base of the sequence shown in SEQ ID NO.1; The third SNP molecular marker is a C / A mutation at the 2250th base of the sequence shown in SEQ ID NO.1.

[0007] Optionally, the genotypes at the site where the first SNP molecular marker is located include CC, TT, and CT; the genotypes at the site where the second SNP molecular marker is located include AA, GG, and AG; and the genotypes at the site where the third SNP molecular marker is located include CC, CA, and AA.

[0008] The present invention also provides a primer pair for detecting the molecular module, comprising an upstream primer with the sequence shown in SEQ ID NO.2 and a downstream primer with the sequence shown in SEQ ID NO.3.

[0009] The present invention also provides the application of the primer pair described above in the genetic breeding of oysters with high temperature tolerance, wherein oysters with the genotype combination of the first SNP molecular marker, the second SNP molecular marker and the third SNP molecular marker at the loci of TT / GG / AA have higher high temperature tolerance than other genotypes.

[0010] The present invention also provides the application of the primer pair described above in the genetic breeding of oysters with high KAT2 protein expression. Oysters with the genotype combination of the first SNP molecular marker, the second SNP molecular marker and the third SNP molecular marker at the loci of TT / GG / AA have higher KAT2 protein expression abundance than other genotypes.

[0011] This invention also provides a method for breeding heat-resistant oysters, comprising the following steps: Using the DNA of the oyster sample to be tested as a template, PCR amplification was performed using the primer pair described above. The genotype of the corresponding site in the molecular module was determined based on the amplification product. Oysters with the dominant gene at the corresponding site were selected as parents for breeding. The dominant genotype at the site of the first SNP molecular marker is TT, the dominant genotype at the site of the second SNP molecular marker is GG, and the dominant genotype at the site of the third SNP molecular marker is AA.

[0012] This invention also provides a method for breeding oysters with high expression of KAT2 protein, comprising the following steps: Using the DNA of the oyster sample to be tested as a template, PCR amplification was performed using the primer pair described above. The genotype of the corresponding site in the molecular module was determined based on the amplification product. Oysters with the dominant gene at the corresponding site were selected as parents for breeding. The dominant genotype at the site of the first SNP molecular marker is TT, the dominant genotype at the site of the second SNP molecular marker is GG, and the dominant genotype at the site of the third SNP molecular marker is AA.

[0013] Optionally, the PCR amplification reaction system consists of 1 μL DNA template, 0.5 μL universal high-fidelity enzyme, 0.5 μL dNTP, 1 μL each of forward and reverse primers, 8.5 μL double-distilled water, and 12.5 μL buffer.

[0014] Optionally, the PCR amplification reaction conditions are: 94℃ for 1-5 min; 94℃ for 10-30 s, 55℃ for 10-30 s, 72℃ for 3 min, 35-40 cycles; 72℃ for 0-10 min.

[0015] Optionally, the genotype determination method includes determination using FastNGS sequencing.

[0016] The present invention discloses the following technical effects: This invention provides a molecular module related to the abundance and heat resistance of the oyster KAT2 protein. Based on population genomics analysis, three highly linked synonymous mutation sites (c.243 C>T, c.507 A>G, and c.801 C>A) were precisely located in the exons of the KAT2 gene coding region. Multiple validations in wild and hybrid populations confirmed the causal relationship between homozygous mutant genotype combinations (TT / GG / AA) and KAT2 protein abundance and global acetylation levels. Compared to the high-cost strategy of genome-wide selection breeding, which relies on massive amounts of sporadic markers, this invention only requires the detection of three functional SNP sites to achieve accurate assessment of oyster heat tolerance, significantly reducing genotyping costs and data analysis complexity. This provides an economical and efficient technical solution for heat-resistant molecular breeding of low-value-added aquaculture species such as oysters.

[0017] This invention validated the effectiveness of the molecular module at the levels of protein expression and individual survival rate through Western blotting and semi-lethal temperature heat stress experiments. The results demonstrated that individuals carrying the dominant TT / GG / AA genotype exhibited significantly higher KAT2 protein expression and survival rates under high-temperature stress compared to other genotypes. Based on this, the developed detection primer pairs and breeding methods can be directly used for non-invasive genotyping of oyster parents, allowing for targeted selection of dominant individuals for breeding, thereby effectively improving the high-temperature resistance of offspring populations. This molecular module overcomes the shortcomings of traditional heat-resistant phenotypic selection, which is highly susceptible to environmental interference and has a long cycle, providing a reliable molecular breeding tool for alleviating large-scale oyster mortality caused by high summer temperatures. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram (A) and a quantitative statistical diagram (B) of KAT2 protein abundance in Pacific oyster and Fujian oyster in Example 1 of the present invention. Figure 2 This is a schematic diagram of the genotyping of three sites c.243 C>T (A), c.507 A>G (B), and c.801 C>A (C) in the molecular module markers of Pacific oyster and Fujian oyster in Example 1 of the present invention; Figure 3 This is a schematic diagram (A) and a quantitative statistical diagram (B) of the KAT2 protein abundance in different genotypes of the F2 hybrid of Pacific oyster and Fujian oyster in Example 2 of the present invention. Figure 4 This is a schematic diagram of the global protein acetylation levels of individuals with different genotypes within the molecular module in Example 2 of the present invention; Figure 5 This is a survival curve of individuals with different genotypes in the molecular module after heat shock at a semi-lethal temperature in the F2 generation of hybridized Pacific oyster and Fujian oyster in Example 3 of the present invention. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The advantage of the molecular module described in this invention in the targeted genetic improvement of oysters lies in its ability to rapidly perform high-throughput, non-destructive genotyping of breeding parent oysters, accurately selecting individuals carrying homozygous dominant heat-resistant molecular modules (TT / GG / AA) as parents, thereby directionally improving the heat tolerance of offspring populations. The molecular module obtained in this invention has undergone repeated functional verification and multiple population-level evaluations in wild populations of *Crassostrea gigas* and *Crassostrea kwangsiensis*, as well as hybrid populations, exhibiting significant differences in heat tolerance. It possesses high reliability and can provide valuable assistance for molecular breeding work on heat-resistant oysters.

[0026] The present invention will now be described in detail with reference to specific embodiments.

[0027] Example 1: Screening and Identification of Genetic Loci in Molecular Modules 1. Acquisition of experimental materials and homogenized breeding Wild long oyster ( C. gigas ) and Fujian oysters ( C. angulata The parent oysters were collected from their natural habitats in Qingdao, Shandong and Xiamen, Fujian. The collected parent oysters were transferred to the Qingdao aquaculture base for artificial domestication until sexual maturity. Subsequently, a first-generation homogeneous aquaculture experiment was conducted on the two species. The specific procedures were as follows: 30 mature female individuals from each of the *Crassostrea gigas* and *Crassostrea kwangsiensis* species were collected, their eggs were thoroughly mixed, and then divided into 30 egg samples. Simultaneously, sperm from 30 mature male individuals from each species was collected and used for targeted artificial insemination with each egg sample of the corresponding species. The fertilized eggs and hatched larvae were cultured according to standardized procedures at a farm in Laizhou, Shandong. Four-month-old F1 individuals were transferred to Muping, Shandong, for further rearing until eight months of age for subsequent experiments.

[0028] 2. Comparison of KAT2 protein abundance under heat stress Eight-month-old populations of *Crassostrea gigas* and *Crassostrea kwangsiensis* were co-exposed to sublethal heat shock at 37°C for 12 hours. Gill tissue samples were collected, flash-frozen in liquid nitrogen, and stored at -80°C. 0.1 g of tissue was weighed, 1 mL of lysis buffer containing protease inhibitors was added, and the mixture was homogenized thoroughly. The homogenate was then centrifuged at 14,000×g at 4°C, and the supernatant was collected. 4× loading buffer was added to the supernatant, and the mixture was heated at 95°C for 10 min to denature the proteins. Protein samples were separated by SDS-PAGE (120 V, 100 mA, 80 min), and the charged protein strips were then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 30 min, incubated with primary antibody overnight at 4°C, washed three times with 1×TBST, and then incubated with secondary antibody at 4°C for 4 h. The incubated membrane was developed using the Omni-ECL™ ultrasensitive chemiluminescence kit and analyzed using Molecular Imager. ® The Gel Doc™ XR System acquires images.

[0029] The results showed that under heat stress, the KAT2 protein abundance in heat-tolerant Fujian oysters remained at a significantly high level, while the KAT2 protein abundance in heat-sensitive Pacific oysters was relatively stable. Figure 1 ).

[0030] 3. Population genomics analysis and molecular module locus identification Whole-genome resequencing data of the two species were extracted, and the genetic differentiation index (F) between the Pacific oyster and the Fujian oyster population was calculated by joint analysis. ST ) and nucleotide diversity ratio (θ) π A population-selective elimination analysis was conducted using ratios. A high linkage disequilibrium (LD) region under strong natural selection was identified in the coding region and flanking regions of the KAT2 gene on chromosome 5. Through fine mapping and significance analysis, three highly differentiated and strongly linked synonymous mutation sites (Chr05: 55,431,891; Chr05: 55,433,105; Chr05: 55,434,090) were finally identified in exons 2, 4, and 6 of the KAT2 coding region. The nucleotide sequences are shown in SEQ ID NO.1. A C / T mutation (c.243 C>T) exists at base 51, an A / G mutation (c.507 A>G) exists at base 1265, and a C / A mutation (c.801 C>A) exists at base 2250.

[0031] SEQ ID NO.1: Where y represents C / T, r represents A / G, and m represents C / A.

[0032] 4. Validation of wild populations of molecular modules Genetic variation sites within molecular modules amplified by PCR in the above-mentioned wild Pacific oyster and Fujian oyster populations were independently examined using FastNGS.

[0033] (1) DNA extraction: Total DNA was extracted from the gill tissue of each individual using the sodium lauryl sulfate method. 1 μL of RNase (0.1 mg / mL) was added, and the RNA was digested in a water bath at 37°C for 30 minutes to obtain the DNA solution.

[0034] (2) Genotyping: PCR amplification was performed using specific primers. The reaction system was as follows: 1 μL DNA template, 0.5 μL universal high-fidelity enzyme, 0.5 μL dNTP, 1 μL each of primers F and R, 8.5 μL double-distilled water, and 12.5 μL buffer. The first 6 exons of the KAT2 coding region of each individual were obtained by amplification.

[0035] The primer sequences are as follows: Forward primer F: 5'-ACAGAGAATTGCAAGTGCAATGG-3' (SEQ ID NO.2); Reverse primer R: 5'-CTAGGAAAATGGGTAAGGACAAGAG-3' (SEQ ID NO.3); The PCR amplification reaction program was as follows: 94℃ for 1-5 min; 94℃ for 10-30 s, 55℃ for 10-30 s, 72℃ for 3 min, 35-40 cycles; 72℃ for 0-10 min.

[0036] (3) The PCR products of the KAT2 coding region of each individual were subjected to FastNGS sequencing. Based on the chi-square test significance analysis, it was confirmed that in the wild Pacific oyster population, the genotype combinations corresponding to the c.243, c.507, and c.801 sites were highly inclined to the non-mutant CC / AA / CC (heat-sensitive molecular module), while in the wild Fujian oyster population, the proportion of mutant homozygous genotype combinations was extremely high (TT / GG / AA; heat-tolerant molecular module). See [link to relevant documentation]. Figure 2 .

[0037] Example 2: Functional Verification of Genetic Loci in Molecular Modules 1. Acquisition of experimental materials In 2024, wild Pacific oysters and Fujian oysters were collected from Qingdao and Xiamen, respectively. Thirty female Pacific oysters and thirty male Fujian oysters were used for population hybridization to obtain the F1 hybrid population. In 2025, 30 sexually mature females and 30 males of the F1 hybrid population were selected for in-population self-pollination to construct the F2 population, which was then cultured along the coast of Qingdao. In 2026, 100 eight-month-old F2 hybrid individuals were exposed to sublethal heat shock at 37°C for 12 hours. Gill tissue samples were collected, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0038] 2. DNA extraction and genotyping Total DNA was extracted from gill tissue of each individual using the sodium lauryl sulfate method. PCR amplification was then performed using specific primers. The PCR products of the KAT2 coding region of each individual were sequenced and genotyped using FastNGS. Among the 95 genotyped individuals, 29 individuals carrying the heat-sensitive molecular module (CC / AA / CC), 27 heterozygous individuals with the genotype combination CT / AG / CA, and 38 individuals carrying the heat-tolerant molecular module (TT / GG / AA) were identified.

[0039] 3. Comparison of KAT2 protein abundance and global acetylation level under heat stress Proteins were extracted from gill tissues of individuals with different genotype combinations, and then Western blotting analysis was performed using KAT2 and panacetylation antibodies.

[0040] The results showed that under heat stress, the expression level and global acetylation level of KAT2 protein in F2 individuals carrying the heat tolerance molecular module (TT / GG / AA) were significantly higher than those in heterozygous individuals, and exhibited an overwhelming abundance advantage over those carrying the heat sensitivity molecular module (CC / AA / CC). Figure 3 and Figure 4 ).

[0041] Example 3: Application and Verification of Molecular Modules in Actual Breeding 1. Collection of experimental materials In 2026, the Changfu hybrid F2 population will be collected in Qingdao and temporarily raised in the laboratory for half a month to eliminate the impact of the environment.

[0042] 2. Oyster anesthesia Hybrid F2 oysters were placed in an anesthetic solution (500g MgCl2 + 5L seawater and 5L freshwater) for 12 hours. After the oysters were anesthetized and opened, a small amount of gill tissue was collected from the live oysters for total DNA extraction.

[0043] 3. DNA extraction and genotyping Genotyping was performed on the live-sampled Changfu hybrid F2 population using the method described in Example 2. One hundred individuals each carrying the heat-sensitive molecular module (CC / AA / CC), heterozygote (CT / AG / CA), and heat-tolerant molecular module (TT / GG / AA) were selected for subsequent experiments.

[0044] 4. 42℃ Semi-lethal heat stress experiment Three hundred oysters were heat-shocked in seawater at a semi-lethal temperature of 42°C for 1 hour, and then returned to seawater at room temperature (15±2°C) for continuous cultivation for 14 days. The number of dead individuals was counted daily.

[0045] The results showed that the survival rate of individuals with the heat-tolerant molecular module (TT / GG / AA) was significantly higher than that of individuals with the heterozygote (CT / AG / CA) and the heat-sensitive molecular module (CC / AA / CC). Figure 5 This study confirms that SNP markers within the KAT2 gene molecular module are highly reliable molecular indicators of oyster heat tolerance and can be applied to oyster heat-tolerant breeding to improve survival rates under high-temperature stress.

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A molecular module associated with the abundance of KAT2 protein in oysters and its high-temperature resistance, characterized in that, The molecular module includes a first SNP molecular marker, a second SNP molecular marker, and a third SNP molecular marker; The first SNP is marked by a C / T mutation at the 51st base of the sequence shown in SEQ ID NO.1; The second SNP molecular marker is an A / G mutation at the 1265th base of the sequence shown in SEQ ID NO.1; The third SNP molecular marker is a C / A mutation at the 2250th base of the sequence shown in SEQ ID NO.

1.

2. The molecular module according to claim 1, characterized in that, The genotypes at the first SNP molecular marker site include CC, TT, and CT; the genotypes at the second SNP molecular marker site include AA, GG, and AG; and the genotypes at the third SNP molecular marker site include CC, CA, and AA.

3. A primer pair for detecting the molecular module of claim 1 or 2, characterized in that, It includes an upstream primer with the sequence shown in SEQ ID NO.2 and a downstream primer with the sequence shown in SEQ ID NO.

3.

4. The application of the primer pair as described in claim 3 in the genetic breeding of oysters with high-temperature tolerance, characterized in that, Oysters with the genotype combination TT / GG / AA at the sites where the first, second, and third SNP molecular markers are located have higher heat tolerance than other genotypes.

5. The application of the primer pair as described in claim 3 in the genetic breeding of oysters with high KAT2 protein expression, characterized in that, Oysters with the genotype combination TT / GG / AA at the sites of the first, second, and third SNP molecular markers showed higher KAT2 protein expression abundance than other genotypes.

6. A method for breeding heat-resistant oysters, characterized in that, Includes the following steps: Using the DNA of the oyster sample to be tested as a template, PCR amplification was performed using the primer pair described in claim 3. The genotype of the corresponding site in the molecular module described in claim 1 or 2 was determined according to the amplification product. Oysters with the dominant gene at the corresponding site were selected as parents for breeding. The dominant genotype at the site of the first SNP molecular marker is TT, the dominant genotype at the site of the second SNP molecular marker is GG, and the dominant genotype at the site of the third SNP molecular marker is AA.

7. A method for breeding oysters with high expression of KAT2 protein, characterized in that, Includes the following steps: Using the DNA of the oyster sample to be tested as a template, PCR amplification was performed using the primer pair described in claim 3. The genotype of the corresponding site in the molecular module described in claim 1 or 2 was determined according to the amplification product. Oysters with the dominant gene at the corresponding site were selected as parents for breeding. The dominant genotype at the site of the first SNP molecular marker is TT, the dominant genotype at the site of the second SNP molecular marker is GG, and the dominant genotype at the site of the third SNP molecular marker is AA.

8. The method according to claim 6 or 7, characterized in that, The PCR amplification reaction system consisted of 1 μL DNA template, 0.5 μL universal high-fidelity enzyme, 0.5 μL dNTPs, 1 μL each of forward and reverse primers, 8.5 μL double-distilled water, and 12.5 μL buffer.

9. The method according to claim 6 or 7, characterized in that, The PCR amplification reaction conditions were: 94℃ for 1-5 min; 94℃ for 10-30 s, 55℃ for 10-30 s, 72℃ for 3 min, for 35-40 cycles; and 72℃ for 0-10 min.

10. The method according to claim 6 or 7, characterized in that, The genotype determination method includes determination using FastNGS sequencing.

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